Abstract Protein-ligand interactions play a pivotal role in fundamental biological processes including cellular signaling and regulation. Experimental screening for interactions with drug candidate compounds and fragments further represents an indispensable step in drug discovery. Traditional methods for determining such interactions often require highly purified proteins, which in particular are not always available in the case of membrane proteins. The same limitations exist for the characterization of functional complexes formed by recruitment of multiple constituents on a membrane or within a cell. Notwithstanding, 60% of current drugs target membrane proteins. Here, a method will be developed for determining interactions between small molecules of arbitrary type, directly with receptors or other components located on the surface or within a cell. Nuclear magnetic resonance (NMR) signals of ligands will be enhanced by several orders of magnitude using dissolution dynamic nuclear polarization (D-DNP). The non-equilibrium spin states produced by this hyperpolarization technique results in sensitivity gains enabling detection to targets in the nanomolar concentration range. At the same time, hyperpolarization provides contrast over complicated background spectra. It thus enables the label- free, selective detection of the ligand of interest, while retaining the chemical information available in NMR spectroscopy. In a first aim of the project, an experimental method for detecting and characterizing binding in heterogeneous models containing proteins in lipid vesicles will be developed. Nuclear spin relaxation under competitive binding, and intra- or interligand nuclear Overhauser effect (NOE) will provide binding affinity measurements and structural constraints within the binding pocket. In a second aim, these methods will be applied to characterize the binding of ligands to a G-protein coupled chemokine receptor on the surface of mammalian cells. A dedicated device for NMR detection of cell cultures perfused with hyperpolarized media will be developed for this purpose. A third aim will extend these methods to access targets within the cytosol or an internal membrane of the cell, here specifically an oligomeric signaling protein from the cGAS-STING interferon induction pathway. Hyperpolarized water will be naturally transported across the cell membrane. Hyperpolarization will then transfer to intracellular components by proton exchange or NOE, resulting in detectable signal changes of unlabeled or selectively isotope labeled ligands within the cell. Together, these aims will provide a comprehensive, novel toolset to characterize protein-ligand interactions in the natural environment of the cell.